| Several aspects of nonlinear chromatography were studied to develop effective preparative HPLC techniques for the separation of proteins and other biologicals. The technique of frontal analysis using a miniaturized liquid chromatograph was employed to obtain isotherms of amino acids, peptides, nucleotides and proteins on a variety of stationary phases commonly used in HPLC. Adsorption of the simpler molecules were studied under a variety of operating conditions to obtain a general understanding about the effect of those parameters on the isotherm. Most of the isotherms exhibited favorable concave downward shapes, although non-Langmuirian isotherms were observed in some cases. The isotherm of the nucleotide adenosine monophosphate was sigmoidal, but by manipulating various operating parameters, the isotherm was converted into a Langmuir form. Since analysis of the complex adsorption behavior could provide useful information about solute-solute interactions, simple thermodynamic models were developed. The models took into account the "stacking" of the molecules in solution and accurately predicted the deviation from the regular adsorption behavior.; Small peptides were found to exhibit crossing Langmuirian isotherms under certain conditions, and the implications of this phenomenon was evaluated through overloaded elution and displacement studies. In overloaded elution, seemingly anomalous chromatograms were observed, and in displacement, a constant mixed zone separation pattern resulted. This behavior was attributed to the formation of an adsorption azeotrope, which implied that the multicomponent isotherms crossed. By manipulating the operating conditions, the isotherms were uncrossed and the nonlinear separations proceeded without complication.; The adsorption characteristics of proteins on a given sorbent were found to be very sensitive to the mobile phase composition. Under conditions of strong binding, the isotherms were rectangular in shape and adsorption was irreversible on the time scale of chromatographic interest. It was shown, however, that these characteristics did not preclude chromatography at high feed concentrations because competition moderated the otherwise irreversible binding and suppressed the multicomponent isotherms. Through a study of reversed phase displacement chromatography of proteins, several interesting phenomena were observed. Frontal chromatography and stepwise desorption were found to be more suitable when the separation factors were sufficiently large. The fractionation of a mixture of human growth hormone was used to demonstrate the efficacy of this separation scheme. It was also shown that strong binding conditions yielded higher selectivity in certain cases, and that small variations in the mobile phase composition gave rise to dramatic changes in adsorption selectivity. |
